RNA PROCESSING FACTOR2 Is Required for 5′ End Processing of nad9 and cox3 mRNAs in Mitochondria of Arabidopsis thaliana  

Abstract

In mitochondria of higher plants, the majority of 59 termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 59 end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein. The functional importance of this mitochondrial restorer of fertility-like protein, designated RNA PROCESSING FACTOR2 (RPF2), is confirmed by the analysis of a respective T-DNA knockout mutant and its functional restoration by in vivo complementation. RPF2 fulfills two functions: it is required for the generation of a distinct 59 terminus of transcripts of subunit 9 of the NADH DEHYDRO-GENASE complex (nad9) and it determines the efficiency of 59 end formation of the mRNAs for subunit 3 of the CYTOCHROME C OXIDASE (cox3), the latter also being influenced by mitochondrial DNA sequences. Accordingly, recombinant RPF2 protein directly binds to a nad9 mRNA fragment in vitro. Two-dimensional gel electrophoresis and immunodetection analyses reveal that altered 59 processing does not influence accumulation of the nad9 and cox3 polypeptides. In accessions C24, Oystese-1, and Yosemite-0, different inactive RPF2 alleles exist, demonstrating the variability of this gene in Arabidopsis. The identification of RPF2 is a major step toward the characterization of 59 mRNA processing in mitochondria of higher plants.

RNA Processing Factor 7 and Polynucleotide Phosphorylase Are Necessary for Processing and Stability of nad2 mRNA in Arabidopsis Mitochondria

“…These factors, exclusively P-class PPR proteins comprising 14 PPR motifs or more, are required for efficient 5′ processing of the major transcript species transcribed from one, two or three different mitochondrial genes, [25][26][27]33 but as found recently by the analysis of the restorer of fertility-like PPR protein 9 (RFL9), are also involved in 5′ processing of rare RNA molecules in particular ecotypes. 38 The relatively high number of PPR motifs present in these proteins allowed to predict the putative binding sites on the RNA targets, which is exclusively found upstream of the 5′ end of the mature transcript.…”

“…24 These include proteins that are similar to restorer of fertility (RF) from other plant species. [25][26][27] RF proteins can rescue cytoplasmic male sterility (CMS), a maternal inherited deficiency to produce or release fertile pollen. [28][29][30][31] Recently CMS in Arabidopsis has been reported, but a nuclear gene restoring male fertility in this system has not been identified.…”

“…26,27 CR-RT-PCR experiments were done according to established protocols. 34 Northern blot analyses were performed with Biodyne A nylon membranes according to protocols given by the manufacturer (Pall Corporation, http://www.pall.com).…”

The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes

“…The first characterized RFL gene corresponded to the RNA Processing Factor 2 (RPF2) and was shown to be responsible for the production of a nad9 mRNA 5' end located 202 nucleotides upstream of the translation initiation codon in Arabidopsis Columbia (Col.0) and Landsberg erecta (Ler) accessions. 53 Knockout mutant analysis further revealed that RPF2 is also in charge of the production of a predominant 5' end for cox3 mRNA in Col.0 and Ler plants, which is located at position -380 upstream of the translation start codon. The analysis of the RPF2 gene in Arabidopsis accessions exhibiting nad9 and cox3 5' ends differing from Col.0 revealed that they contained point-mutated or highly rearranged versions of the RPF2 gene.…”

Insights into PPR Gene Family in Cajanus Cajan and Other Legume Species

“…By using classical genetic screens, number of PPR mutants have been characterized with varied phenotypes ranging from those showing photosynthetic defect [5] to restricted growth [6], defective seed and embryo development [7], aberrant leaf growth [8] and restoration of pollen fertility [9]; implying the role of PPRs as sequence specific RNA binding proteins in organelles. Other reports also suggest important role of PPR and these includes, abnormal splicing of chloroplast targeted PPR encoding Rpl2 gene in rice resulted in mutant with white stripe leaf (WSL mutant) characterized by enhanced sensitivity to abiotic stresses and chlorotic striations during its early development [10], Rf1A in rice functions in atp6 mRNA editing [11], RPF2 affects mitochondrial nad9 and cox3 mRNAs in arabidopsis [12] and so on. Non plant organisms have very few PPRs whereas great expansion of this gene family via retrotransposition has been observed in plants [13].…”